Although non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic triglyceride (TG) accumulation, its pathogenesis remains poorly understood. The ?-Klotho (Klb) gene encodes an obligatory co-receptor necessary for biological functions of FGF21, which promotes fatty acid oxidation in liver, thereby ameliorating hepatic steatosis. However, fatty liver is paradoxically associated with increased FGF21 levels in both obese humans and mice, suggesting a FGF21-resistant state where increased FGF21 fails to block the development of fatty liver. The mechanism underlying FGF21 resistance remains elusive. Epigenetic regulation, including DNA methylation, is a molecular link between environmental factors (e.g. diets) and complex diseases (e.g. obesity and NAFLD). Our preliminary data suggested that DNA methylation, regulated by DNA methyltransferases (DNMTs), is an important determinant of hepatic lipid metabolism. Upregulation of DNMTs by saturated fatty acids (SFAs) and the pro-inflammatory cytokines may contribute to deregulated hepatic lipid metabolism and fatty liver in obesity. Moreover, our genome-wide profiling of DNA methylation using reduced representation bisulfite sequencing (RRBS) analysis revealed a significant increase in DNA methylation at the Klb promoter in liver of diet-induced obese (DIO) mice, which is associated with downregulation of Klb expression in RNA-seq analysis. The downregulation of Klb expression may limit the ability of increased FGF21 to promote hepatic lipid oxidation, leading to FGF21 resistance. We therefore hypothesize that epigenetic programming of the Klb promoter by nutritional cues (e.g. SFAs) and pro-inflammatory stimuli (e.g. cytokines), levels of which are commonly elevated in obesity, mediates hepatic lipid accumulation by downregulating the FGF21 pathway, leading to fatty liver in obesity. Aim 1 will determine whether enhanced DNA methylation at the Klb promoter by SFAs and cytokines impairs hepatic FGF21 signaling and lipid metabolism, leading to hepatic TG accumulation. We will first test the hypothesis that DNMT1 mediates the enhanced DNA methylation at the Klb promoter by SFAs and cytokines, leading to hepatic FGF21 resistance and lipid accumulation. We will then determine the direct role of Klb promoter methylation in hepatic FGF21 signaling and lipid metabolism using a novel CRISPR/RNA-guided system to specifically induce methylation/demethylation at the Klb promoter. Aim 2 will determine whether liver-specific inhibition of DNA methylation promotes hepatic FGF21 signaling and ameliorates fatty liver in genetic mouse models fed high fat diet. We have generated liver-specific DNMT1 and 3a knockout (LD1KO and LD3aKO) mice by crossing DNMT1- and 3a-floxed mice with Albumin-Cre mice. We will further determine FGF21 signaling, hepatic lipid metabolism and TG accumulation in LD1KO and LD3aKO mice fed HF diet. We have also established a Cre-dependent CRISPR/RNA-guided approach for targeted DNA methylation/demethylation at the Klb promoter specifically in liver of mice to test the hypothesis that enhanced DNA methylation at the Klb promoter in hepatocytes mediates impaired FGF21signaling and lipid metabolism.